Chromatography Forum

Hi,

I am looking for UPLC method to determine Organic acids: citric, malic, tartaric, and phosphoric acid.
Can any experties on UPLC help me?

I found one method from Waters website, but this method is valid with UV detector?

Water website method is:

Aquity BEH Amide, 2.1X 100mm, 1.7uL Column
Mobile Phase A: 50/50 MeCN/H2O with 10mM CH3COONH4, pH9
Mobile Phase B: 95/5 MeCN/5 MeCN/H2O iwth 10mM CH3COONH4, pH9
Instrument: UPLC and TQD

Please help me~~

Thanks,

Kelly

Which organic acids?

Hi,
the mobile phase in the HILIC method you are looking at is ammonium acetate, pH 9, just 10 mM. In principle it is compatible with UV detection, but it is not very UV transparent at short wavelengths, say below 230 nm. If your organic acids are detectable at 254 nm or higher, you are OK. I suspect that they are not, they may not contain phenyl or other UV detectable moieties. That is why you intend to use HILIC stationary phase, right? To achieve reasonable retention for hydrophilic compounds? Anyway, the solution could be to replace the buffer with another, say good old phosphate adjusted to pH ~7 - I suspect that organic acids have to be charged to have good HILIC retention. Such buffer should be UV transparent. Stay low with concentration of buffer, few mM will do it. Double check that your buffer is soluble in your mobile phase. "Organic mobile phase" should contain ~5-10% water, otherwise you will start precipitating your buffer.

I consulted with my colleagues. For the acids you mentioned (minus phosphoric) it is possible to use HILIC in isocratic mode with mobile phase consisting of 0.1% phosphoric acid and high ACN content. UV detection at 210 nm. Obviously, phosphate cannot be detected in this case.

Alternatively, you can try CSH C18 column with 0.1% phosphoric acid as mobile phase. Retention in RP mode is probably aided by residual charge on CSH column surface. Conditions: ACQUITY CSH C18, 2.1 x 150 mm, 1.7 μm, Part Number 186005298, Mobile phase: 0.1% H3PO4 in H2O, Isocratic flow rate: 0.2 mL/min, column temperature ~25 C, flow rate 0.2 ml/min or faster. Tartaric acid, Malic acid, Lactic Acid, Acetic Acid, Succinic Acid, and Citric Acids were resolved (listed in order of elution) within 7 minutes.

except for phosphoric acid, i know you can do all the other ones using a C-18 column that can run with a 100% buffer, the AQs types, and also with a c-18 with a polar embedded group that is as well 100% buffer proof.
the main problem here is that these applications run with a phosphoric acid buffer/solution.

Thank you so much every one!

To Martin,
Finally, I will start using UPLC to analyze organic acid except phosphoric acid.
I will take your advise and I want to try the method you suggested.

Thanks!!

Kelly

to add another suggestion

there is also one application note in the Waters Atlantis application notebook.
(720000472EN)

Atlantis T3 column with 20 mM NaH2PO4 at pH 2.7, T=30°C, 0.5 ml/min at 4.6x150mm column, detection @ 210 nm

(personaly proofed to be working )

On UPLC, I would try the HSS T3 as the most similar (don't know the above mentioned Amide or CSH column yet)

Thanks!

Oh, good luck! What is the range of your acids and what matrix are they present in? I was trying a similar system, but at 210 nm is difficult to use gradient: baseline issues (As I reported and asked for help ). How about a precolumn derivatization with Phenacyl Bormide? (Daniel Knapp ADerivatization book, pg 187).
Pls update us about your progress.

I have another question on a similar topic: any suggestions for direct analysis of lactic acid and capric,and caproic acids? The product is dissolved in water (with the aid of some surfactants, of course). I tried direct injections at 210nm, but due to differences in the acids I need to use gradient, and at 210nm is not so good. Thinking of doing precolumn derivatization, as I mentioned above, but would gladly do it without the extra step. It is for an industrial, fast paced QA application. Any suggestions is a big help, thank you.

Hi everyone,

I tried the Atlantis T3 column. My analytes are coeluting.
Tartaric acid, Malic acid, lactic acid, Acetic acid, and citric acid.

Base line seems to be fine, but they all eluted in 1min with 0.5mL/ min and 3min with 0.1mL/min.

I guess I need to optimize the method.
Any suggestions, please!

Thanks,

Kelly

?

what colum dimension?
Did you condition your column with 100% of organic first, then change to the
mobile phase?
Seems your compounds are eluting close or even befor t0?
Also the retention times for 0.1 ml/min should be 5 times the ones of 0.5ml/min.
Is your flow correct?

I worked on 4.6x100mm, 3µm, no modifications needed, but worked on T=35°C.

Retention factors observed (about):
Oxalic 0.11
Tartaric 0.30
Malic 0.79
Ascorbic 1.12
Lactic 1.3
Acetic 1.51
Citric 2.51
Succinic 3.55
Propionic 6.0

What are the conditions for this analysis: solvents, gradinet, flow, temperature?

Bio-Rad, Dionex, et al also have organic acids analysis columns. These are generally cation exchange resins in hydrogen form and work by a mixed mechanism of ion exclusion and partition. Mobile phase is isocratic, usually dilute sulfuric acid.